Neuropharmacology 148 (2019) 320e331

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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

N-Oleoyl-glycine reduces nicotine reward and withdrawal in mice

Giulia Donvito a, 1, Fabiana Piscitelli b, 1, Pretal Muldoon a, Asti Jackson a,
Rosa Maria Vitale b, Enrico D'Aniello b, f, Catia Giordano c,
Bogna M. Ignatowska-Jankowska a, Mohammed A. Mustafa a, Francesca Guida c,
Gavin N. Petrie d, Linda Parker d, Reem Smoum e, Laura Sim-Selley a, Sabatino Maione c,
Aron H. Lichtman a, g, *, M. Imad Damaj a, **, Vincenzo Di Marzo b, ***,
Raphael Mechoulam e
a
Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA, USA
b
Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Naples, Italy
c
Endocannabinoid Research Group, Department of Experimental Medicine, Section of Pharmacology, Second University of Naples, Naples, Italy
d
Department of Psychology and Collaborative Neuroscience Graduate Program, University of Guelph, Guelph, ON, Canada
e
Institute for Drug Research, Medical Faculty, Hebrew University, Jerusalem, Israel
f
Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Naples, Italy
g
Department of Medicinal Chemistry, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA, USA

a r t i c l e i n f o a b s t r a c t

Article history: Cigarette smokers with brain damage involving the insular cortex display cessation of tobacco smoking,
Received 6 September 2017 suggesting that this region may contribute to nicotine addiction. In the present study, we speculated that
Received in revised form molecules in the insular cortex that are sensitive to experimental traumatic brain injury (TBI) in mice
1 March 2018
might provide leads to ameliorate nicotine addiction. Using targeted lipidomics, we found that TBI eli-
Accepted 17 March 2018
cited substantial increases of a largely uncharacterized lipid, N-acyl-glycine, N-oleoyl-glycine (OlGly), in
Available online 19 March 2018
the insular cortex of mice. We then evaluated whether intraperitoneal administration of OlGly would
alter withdrawal responses in nicotine-dependent mice as well as the rewarding effects of nicotine, as
Keywords:
N-oleoyl glycine
assessed in the conditioned place preference paradigm (CPP). Systemic administration of OlGly reduced
Nicotine withdrawal mecamylamine-precipitated withdrawal responses in nicotine-dependent mice and prevented nicotine
Cannabinoid receptor-1 (CB1) CPP. However, OlGly did not affect morphine CPP, demonstrating a degree of selectivity. Our respective
Conditioned place preference (CPP) in vitro and in vivo observations that OlGly activated peroxisome proliferator-activated receptor alpha
Insular cortex (PPAR-a) and the PPAR-a antagonist GW6471 prevented the OlGly-induced reduction of nicotine CPP in
Peroxisome proliferator-activated receptor mice suggests that this lipid acts as a functional PPAR-a agonist to attenuate nicotine reward. These
alpha (PPAR-a) findings raise the possibility that the long chain fatty acid amide OlGly may possess efficacy in treating
nicotine addiction.
© 2018 Published by Elsevier Ltd.

1. Introduction

* Corresponding author. Department of Pharmacology and Toxicology, Virginia
Commonwealth University, PO Box 980613, Kontos Medical Sciences Building, 1217
East Marshall Street, Richmond, VA, 23298, USA.
** Corresponding author. Department of Pharmacology and Toxicology, Virginia
Commonwealth University, PO Box 980613, Kontos Medical Sciences Building, 1217
East Marshall Street, Richmond, VA, 23298, USA.
*** Corresponding author. Endocannabinoid Research Group, Institute of Bio-
molecular Chemistry, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34,
Comprensorio Olivetti, 80078, Pozzuoli, Napoli, Italy.
E-mail addresses: alichtma@vcu.edu (A.H. Lichtman), m.damaj@vcuhealth.org
(M.I. Damaj), vdimarzo@icb.cnr.it (V. Di Marzo).
1
These authors contributed equally to this work.
Dependence to tobacco represents one of the most frequent
causes of mortality and morbidity in the world (Doll et al., 2004).
Nicotine, a primary constituent of tobacco responsible for its
rewarding effects, acts on nicotinic acetylcholine receptors
(nAChRs), which are expressed on pre- and post-synaptic terminals
(Albuquerque et al., 2009) in a variety of CNS pathways, including
the mesolimbic reward system (Watkins et al., 2000). Chronic to-
bacco use often induces dependence and cessation can lead to af-
fective (e.g., anxiety, anhedonia, depression, dysphoria,
hyperalgesia, and irritability), somatic (e.g., tremors, bradycardia,

https://doi.org/10.1016/j.neuropharm.2018.03.020
0028-3908/© 2018 Published by Elsevier Ltd.
 G. Donvito et al. / Neuropharmacology 148 (2019) 320e331
gastrointestinal discomfort, and increased appetite), and cognitive
(e.g., difficulty concentrating and impaired memory) withdrawal
symptoms (Heishman et al., 2010). Naqvi and co-workers (Naqvi
et al., 2007) reported that smokers with brain damage involving
the insula, a region implicated in conscious urges, were more likely
than smokers with brain damage not involving the insula to un-
dergo a disruption of smoking addiction. Although the neurobio-
logical mechanisms underlying this association remain to be
determined, preclinical data also implicate a role of the insular
cortex in nicotine reward. Specifically, rats given long access to
nicotine self-administration had increased levels of dopamine and
cAMP-regulated phosphoprotein of 32 kD phosphorylated at the
protein kinase A site in the insular cortex (Abdolahi et al., 2010).
This observation along with human data (Naqvi et al., 2014) suggest
a role of this neuroanatomical region in addictive behavior and that
neurochemicals within the insular cortex that are sensitive to brain
injury may modulate nicotine addiction.
As a starting point to test molecules that may be useful to treat
nicotine addiction, we sought to identify neurochemicals in the
insular cortex of mice that may be altered by traumatic brain injury
(TBI). Twenty-four h after TBI, we harvested insular cortex as well
as comparison regions (i.e., hippocampus, and hypothalamus) for
analysis. We initially focused on endocannabinoids, because of
their well-known involvement in nicotine reward and withdrawal
signs in nicotine-dependent laboratory animals (Castan ~e
 et al.,
2005, 2002; Gamaleddin et al., 2015; Le Foll and Goldberg, 2004;
Merritt et al., 2008; Valjent et al., 2002), as well as related lipid
signaling molecules. Among several investigated lipids, in addition
to the endocannabinoids N-arachidonoylethanolamine (ananda-
mide; AEA) (Devane et al., 1992) and 2-arachidonylglycerol (2-AG)
(Mechoulam et al., 1995; Sugiura et al., 1995), we examined N-
acylethanolamines, N-acyldopamines, N-acylserines and N-
acylglycines.
Based on the results of the lipidomic analyses of TBI and control
brain areas, we selected a largely uncharacterized member of the
latter class, N-oleoyl glycine (OlGly) (Bradshaw et al., 2009). Spe-
cifically, we tested whether exogenous administration of this
molecule would attenuate precipitated withdrawal responses in
nicotine-dependent mice (Jackson et al., 2008). Additionally, we
used the conditioned place preference paradigm (CPP) to infer
whether it would attenuate the rewarding effects of nicotine (Kota
et al., 2007). In order to elucidate potential underlying mechanisms
of action of OlGly, we examined two likely targets. First, we tested
whether it interacts with the endocannabinoid system. Specifically,
we investigated if it binds to human recombinant CB1 and CB2 re-
ceptors transfected on HEK-293 cells, inhibits the primary AEA
hydrolytic enzyme fatty acid amide hydrolase (FAAH), or elicits
in vivo pharmacological effects, as assessed in the tetrad assay,
which is highly sensitive to CB1 receptor agonists (Little et al., 1988;
Martin et al., 1991). Second, we examined whether it binds and
produces its pharmacological effects through peroxisome
proliferator-activated receptor a (PPAR-a), which has been
demonstrated to play a role in nicotine dependence (Jackson et al.,
2017; Mascia et al., 2011; Panlilio et al., 2012).
2. Material and methods
2.1. Subjects
Male ICR mice (6e8 weeks old; Harlan, Indianapolis, IN) with a
body mass of 27e32 g served as subjects in all in vivo pharmacology
experiments. Mice were group-housed (four per cage) for at least
one week before the beginning of experiments on a 12/12 light/
dark cycle (lights on at 0600 h), with an ambient temperature of
20e22
C and humidity of 55e60%. Standard rodent chow and tap
321
water were available ad libitum. Male C57BL/6 mice (Charles River,
Italy) weighing 18e20 g were used for the mild TBI Weight Drop
(WD) model.
All animal protocols were approved by the Virginia Common-
wealth University Institutional Animal Care and Use Committee,
were in accordance with the National Institutes of Health Guide for
the Care and Use of Laboratory Animals and with the National In-
stitutes of Health Guide for the Care and Use of Laboratory Animals,
and by the Animal Ethics Committee of The Second University of
Naples, in compliance with Italian (D.L. 116/92) and European
Commission (O.J. of E.C. L358/1 18/12/86) regulations on the pro-
tection of laboratory animals.
2.2. Synthesis of OlGly
To a solution of oleic acid (1 gm, 3.54 mmol) and N,N-dime-
thylformamide (266 mL, 3.64 mmol) in dry methylene chloride
(10 mL) was added dropwise oxalyl chloride (2.0 M solution in
methylene chloride, 3.5 mL, 7 mmol) under nitrogen atmosphere.
The reaction mixture was stirred for 1 h and then the solvent was
evaporated under a nitrogen flow. The crude material in methylene
chloride (10 mL) was added to a solution of glycine (800 mg,
10.62 mmol) and 2 N potassium hydroxide in an ice bath. Then, the
reaction mixture was stirred for 1 h, water (10 mL) was added, and
the mixture was acidified to pH 3 with 1 N HCl. The product was
extracted with ether (3  50 mL) and dried (MgSO4), and solvent
was evaporated under reduced pressure.
The crude material was chromatographed on silica gel (eluting
with chloroform: methanol) to yield a crystalline solid. Melting
point 93e94
C (degradation); LC-MS: (M-H)þ ¼ 339 m/z; NMR
(CD3OH, ppm): 5.35e5.32 (m, 2H), 4.45 (s, 2H), 2.13e2.18 (m, 6H),
1.58 (m, 2H), 1.32e1.29 (m, 20H), 0.88 (t, 3H).
2.3. Drugs
[2H]8AEA, [2H]52-AG, [2H]4 PEA, [2H]4 OEA, [2H]8 N-arach-
idonoyldopamine (NADA), [2H]8AraSer and [2H]8AraGly were pur-
chased from Cayman Chemicals (MI, USA). OlGly was synthetized in
the Mechoulam laboratory. The PPAR-a receptor antagonist
GW6471 [N-((2S)-2-(((1Z)-1-methyl-3-oxo-3-(4-(trifluoromethyl)
phenyl)prop-1-enyl)amino)-3-(4-(2-(5-methyl2phenyl-1,3-oxazol-
4-yl) ethoxy)phenyl)propyl)propanamide] was purchased from
tocris (Minneapolis, MN).
CP55,940 (()-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)
phenyl]-trans-4-(3-hydroxypropyl)-cyclohexanol) and morphine
sulfate were generously provided by NIDA (Rockville, MD). OlGly,
CP55,940 and GW6471 were dissolved in a vehicle solution con-
sisting of ethanol (5% of total volume), alkamuls-620 (Sanofi-
Aventis, Bridgewater, NJ) (5% of total volume), and saline (0.9%
NaCl) (90% of total volume). OlGly and CP55,940 were given via the
intraperitoneal (i.p.) route of administration in a volume of 10 ml/
kg ()-Nicotine hydrogen tartrate [()-1-methyl-2-(3-pyridyl)
pyrrolidine (þ)-bitartrate] and mecamylamine HCl were purchased
from Sigma-Aldrich Inc. (St. Louis, MO, USA). Morphine sulfate
[morphine hemi[sulfate pentahydrate]] Nicotine and mecamyl-
amine (2 mg/kg) were dissolved in physiological saline and given
via the subcutaneous (s.c.) route of administration in a volume of
10 ml/kg. For the nicotine CPP study, 0.5 mg/kg nicotine dose was
used because this dose reliably produces significant CPP in ICR
mouse (Kota et al., 2007). Morphine CPP was performed with
10 mg/kg (s.c.) as recently described (Alajaji et al., 2016). For nico-
tine withdrawal studies, 24 mg/kg/day nicotine or saline was
continuously perfused for 14 days using s.c. Osmotic minipumps
(model 2002; Alzet Corporation, Cupertino, CA) that were
implanted under isoflurane anesthesia. This prolonged nicotine
322 G. Donvito et al. / Neuropharmacology 148 (2019) 320e331
administration regimen reliably produces significant withdrawal
syndrome in the three behavioral paradigms used here (Jackson
et al., 2009).
2.4. Surgical preparation and brain injury (mouse WD model)
Experimental mild TBI (TBI) was performed using a weight-drop
device developed in the Naples laboratory. Mice were anesthetized
with intraperitoneal injection of 250 mg/kg Avertin before being
subjected to TBI. After a midline longitudinal incision, the skull was
exposed to locate the area of impact and placed under a metal tube
device where the opening was positioned directly over the animal's
head. The injury was induced by dropping a cylindrical metal
weight (50 g), through a vertical metal guide tube from a height of
20 cm. The point of impact was between the anterior coronal suture
(bregma) and posterior coronal suture (lambda). Immediately
following injury, the skin was closed with surgical wound clips and
mice were placed back in their cages to allow for recovery from the
anesthesia and TBI. Sham mice were submitted to the same pro-
cedure as described for TBI, but without release of the weight.
2.5. Extraction and quantification of endocannabinoids, N-
acylethanolamines, N-acyldopamines, N-acylserines and N-
acylglycines
Brain tissues were rapidly frozen, dounce-homogenized, and
extracted with chloroform/methanol/Tris-HCl 50 mM pH 7.5 (2:1:1,
v/v) containing internal deuterated standards for AEA, 2-AG, PEA,
OEA, NADA, AraSer and AraGly quantification by isotope dilution
(5 pmol for [2H]8AEA; 50 pmol for [2H]52-AG, [2H]4 PEA and [2H]4
OEA; 10 pmol for [2H]8 NADA, [2H]8AraSer and [2H]8AraGly). Then
the lipid extract was purified by open bed chromatography on sil-
ica. Fractions were eluted within increasing amounts of CH3OH in
CHCl3 and part of the 9:1 (v/v) fraction was analyzed by liquid
chromatography-atmospheric pressure chemical ionization-single
quadrupole mass spectrometry for AEA, 2-AG, PEA and OEA
levels, as previously described (Bisogno et al., 2009; Piscitelli et al.,
2011). AEA, 2-AG, PEA and OEA levels were calculated on the basis
of their area ratio with the internal deuterated standard signal
areas. Part of the 9:1 fraction was used for N-acyldopamine iden-
tification, whereas the 7:3 fraction was used for N-acylglycine and
N-acylserine identification and quantification by LC-MS-IT-TOF
(Shimadzu Corporation, Kyoto, Japan) equipped with an ESI inter-
face, using multiple reaction monitoring (MRM). The method for
NADA was as previously described (Bisogno et al., 2009). Quanti-
fication was performed by isotope dilution by using m/z values of
370.3192 and 362.2692 corresponding to the molecular ion
[MþH]þ for deuterated and undeuterated AraGly; or m/z values of
400.3297 and 392.2795 corresponding to the molecular ion
[MþH]þ for deuterated and undeuterated AraSer The recovery of
AraGly and AraSer from rat brain tissues using the extraction and
analytical procedure reported here (see Methods) was 49.1 ± 15.7%
and 42.1 ± 15.9% (n ¼ 7). The LC-ESI-IT-ToF method was specific and
exhibited a limit of detection (LOD, defined as the concentration at
which the signal/noise ratio is greater than 3:1) of 50 fmol in the
MS mode, and 1 pmol in the MS/MS mode for all the compounds
analyzed. Moreover, the ratio between the [MþH]þ peak areas of
undeuterated (0.025e10 pmol) vs. deuterated (1 pmol) AraGly and
AraSer varied linearly with the amount of the respective deuterated
standards. The quantification limit of compounds was 100 fmol and
the reproducibility of the method was 95%e99%. The chromato-
grams of the high-resolution [M þ H]þ values were extracted and
used for calibration and quantification. LC analysis was performed
in the isocratic mode using a Kinetex C18 Column (10 cm  2.1 mm,
5 mm) and CH3OH/water/acetic acid (85:15:0.1 by vol.) as the
mobile phase with a flow rate of 0.15 ml/min. Identification of N-
acyldopamines, N-acylglycines and N-acylserines was carried out
using ESI ionization in the positive mode with nebulizing gas flow
of 1.5 ml/min and curved desolvation line temperature of 250
C.
2.6. Nicotine precipitated withdrawal studies
The minipumps were subcutaneously implanted in mice under
isoflurane anesthesia. The pumps delivered 24 mg/kg/day nicotine
or saline for 14 days. On the morning of day 15 (defined as 0 min),
all mice that received nicotine were given an injection of OlGly (10,
30, or 60 mg/kg, i.p.) or vehicle (time: 0 min). At 15 min, each
mouse was given an s.c. injection of the non-selective nicotinic
acetylcholine receptor (nAChR) antagonist mecamylamine (2 mg/
kg, s.c.). At 25 min, an experimenter, blinded to drug treatment,
evaluated the mice in the elevated plus maze and then for physical
(somatic) nicotine withdrawal signs, as previously described
(Jackson et al., 2008). The mice were first evaluated for 5 min in the
plus maze test in which the duration of time spent on the open
arms versus closed arms of the plus maze was assessed. The
number of arm crosses between the open and closed arms was also
counted as a measure of locomotor activity. Immediately following
plus maze assessment, each mouse was placed in a clear activity
cage without bedding for a 20 min observation period of somatic
signs measured that included paw and body tremors, head shakes,
backing, jumps, curls, and ptosis. The total number of somatic signs
was tallied for each mouse and the average number of somatic signs
during the observation period was plotted for each test group. This
testing sequence was chosen based on our prior studies showing
that this order of testing reduced within-group variability and
produced consistent results (Jackson et al., 2008). An observer
blinded to experimental treatment performed all studies.
In order to test whether OlGly is brain penetrant, ICR mice
received a single administration of OlGly (60 mg/kg, i.p.), or vehicle.
After 15 min, plasma, insular cortex, hippocampus and hypothala-
mus were harvested and processed for the extraction and quanti-
fication of the levels of OlGl,y as described above. The dose of OlGly
used in this experiment and the time point for tissue collection
were based on the behavioral experiments.
2.7. Conditioned place preference (CPP) studies
An unbiased CPP paradigm was performed, as previously
described (Kota et al., 2007). Briefly, the CPP apparatus consisted of
three chambers in a linear arrangement (MedAssociates, St. Albans,
VT, ENV3013) with white and black chambers (20  20  20 cm
each), which also differed in floor texture (white mesh or black
rod). These chambers were separated by a small gray chamber with
a smooth PVC floor. Partitions could be removed to allow access
from the gray chamber to the black and white chambers. On day 1,
animals were confined to the middle chamber for a 5-min habit-
uation period and then allowed to move freely among all three
chambers for 15 min. Time spent in each chamber was recorded,
and no systematic bias was observed in baseline chamber prefer-
ence. Twenty-min conditioning sessions occurred twice a day (days
2e4). During conditioning sessions, mice were confined to one of
the larger chambers. The control group received saline in one large
chamber in the morning and saline in the other large chamber in
the afternoon. The nicotine group received nicotine in one large
chamber and saline in the other large chamber. Treatments were
counterbalanced equally in order to ensure that some mice
received nicotine in the morning while others received it in the
afternoon. The nicotine-paired chamber was randomized among
subjects. Sessions were 4 h apart and were conducted by the same
investigator. On each of the conditioning days, mice were
 G. Donvito et al. / Neuropharmacology 148 (2019) 320e331
pretreated with OlGly (i.p.) or vehicle 15 min prior to nicotine or
morphine (s.c.) injection. Five min after nicotine administration
(0.5 mg/kg, s.c.), subjects were given 20-min conditioning sessions.
In the morphine CPP comparison study, mice were given 30 min
conditioning sessions following a 15 min pretreatment of morphine
(10 mg/kg, s.c.) (Alajaji et al., 2016). In the PPAR-a antagonism
study, GW6471 (2 mg/kg, i.p.) was injected 30 min before OlGly. On
test day (day 5), mice were allowed access to all chambers for
15 min in a drug free state. The preference score was calculated by
determining the difference between the time spent in the drug
paired side during test day versus the time in drug paired side
during the baseline day.
2.8. Tetrad behavioral assessment
Mice were acclimated to the test environment for at least 1 h
prior to testing for tetrad components: spontaneous activity, cata-
lepsy, antinociception, and hypothermia (Little et al., 1988; Martin
et al., 1991; Wiley and Martin, 2003). Mice were assessed for
baseline tail withdrawal latencies and body temperature, given an
intraperitoneal (i.p.) injection of vehicle or drug (OlGly), and 30 min
later assessed in the following order: catalepsy, tail withdrawal test,
and body temperature.
In the locomotor studies, subjects were administered vehicle or
drug and 5 min later were placed into clear acrylic boxes (approx.
44.5 cm  22.25 cm  20.0 cm) contained within sound-
attenuating cabinets equipped with an LED light source and fans
for general air circulation and creation of white noise. The distance
traveled was expressed in cm and the time spent immobile in s
collected and recorded for 10 min using Fire-i™ digital cameras
purchased from Unibrain (San Ramon, CA, USA) and ANY-maze™
video tracking software purchased from Stoelting Company (Wood
Dale, IL, USA). Catalepsy was assessed in the bar test, and the
dependent measure was expressed as immobility time during a
60 s observation period (Ignatowska-Jankowska et al., 2015). Anti-
nociception data were transformed to represent a maximum
percent effect (%MPE) by the following formula: %MPE ¼ [(test
latencypretreatment latency)/(10pretreatment latency)]  100.
Body temperature data were expressed as a difference between the
values collected before ad after the drug or vehicle administration
(D temperature,
C).
2.9. Cumulative CP55,940 dose-response study
This experiment examined whether OlGly would shift the cu-
mulative dose-response curves of CP55,940 in producing catalepsy,
antinociception, and catalepsy, an in vivo assay sensitive to CB1
receptor allosteric modulators (Ignatowska-Jankowska et al., 2015).
Mice were pretreated with either OlGly (60 mg/kg i.p.) or vehicle
10 min before they received the first dose of CP55,940 followed by
each subsequent dose every 40 min. Measurements for catalepsy, in
(bar test), tail-flick, and rectal temperature were taken 30 min
following each CP55,940 administration, as well as prior to any
injections to determine baseline responses. Cumulative doses of
CP55,940 were 0.3, 1, and 3 mg/kg i.p. Locomotor activity was not
assessed due to habituation effects that occur following repeated
testing.
2.10. Cannabinoid receptor binding
Membranes from HEK-293 cells stably transfected with the
human recombinant CB1 receptor (Bmax ¼ 2.5 pmol/mg protein)
and human recombinant CB2 receptor (Bmax ¼ 4.7 pmol/mg pro-
tein) were incubated with [3H]-CP-55,940 (0.14 nM/kd ¼ 0.18 nM
and 0.084 nM/kd ¼ 0.31 nM respectively for CB1 and CB2 receptor)
323
as the high affinity ligand and displaced with 10 mM WIN 55212-2
as the heterologous competitor for nonspecific binding (Ki values:
9.2 nM and 2.1 nM respectively for CB1 and CB2 receptor). OlGly was
tested following the procedure described by the manufacturer
(Perkin Elmer, Italia). Displacement curves were generated by
incubating drugs with [3H]-CP-55,940 for 90 min at 30
C. OlGly
effect on AEA hydrolysis by rat brain membranes, which express
high levels of fatty acid amide hydrolase, were assayed as previ-
ously described (Ortar et al., 2007).
2.11. Molecular modelling studies on OlGly
Starting ligand geometry was built with Ghemical 2.99.2
(Hassinen and Pera €kyla
€, 2001), followed by energy minimization
(EM) at molecular mechanics level first, using Tripos 5.2 force field
parametrization, and then at AM1 semi-empirical level. OlGly was
fully optimized using GAMESS program at the Hartree-Fock level
with STO-3G basis set to derive the partial atomic charges using the
RESP procedure of restrained fit to the HF/6-31G*/STO-3G electro-
static potential. Docking studies were performed with AutoDock
4.2 (Morris et al., 2009). The crystallographic structure of PPAR-a
(PDB entry 2P54) and the ligand were processed with AutoDock
Tools (ADT) package version 1.5.6rc1 (Morris et al., 2009) to merge
non polar hydrogens, calculate Gasteiger charges and select rotat-
able side-chain bonds. Grid for docking evaluation with a spacing of
0.375 Å and 50  50  70 points, centered in the ligand binding
pocket, was generated using the program AutoGrid 4.2 included in
Autodock 4.2 distribution. A 100 molecular docking run was per-
formed adopting a Lamarckian Genetic Algorithm (LGA) and the
following associated parameters: 100 individuals in a population
with a maximum of 15 million energy evaluations and a maximum
of 37000 generations, followed by 300 iterations of Solis and Wets
local search. The docking results was subjected to visual inspection
and as representative binding pose was selected the one with most
favorable binding energy for the subsequent MD simulations of
ligand-PPAR-a complex. The complex was completed by addition of
all hydrogen atoms and underwent EM and then MD simulations
with Amber12 pmemd.cuda module (Go € tz et al., 2012), using ff12SB
version of AMBER force field (Case et al., 2012) for the protein and
gaff parameters for the ligand.
To perform molecular dynamics (MD) simulation in solvent, the
complex was confined in TIP3P water periodic box exhibiting a
minimum distance between solute and box surfaces of 10 Å, using
the tleap module of AmberTools12 program (Wang et al., 2004).
The system was then neutralized by addition of counterions (Naþ)
and underwent 1000 steps of EM with solute atoms harmonically
restrained to their starting positions using a force constant of
10 kcal mol-1Å-1. The solvated complex was submitted to 90 ps
restrained MD (5 kcal mol-1Å-1) at constant volume, gradually
heating the system to 300 K, followed by 60 ps restrained MD
(5 kcal mol-1Å-1) at constant temperature (300 K) and pressure
(1 atm) to adjust system density. Production MD simulation was
carried out at constant temperature (300 K) and pressure (1 atm)
for 50 ns with a time-step of 2 fs. Bonds involving hydrogens were
constrained using the SHAKE algorithm (Ryckaert et al., 1977).
2.12. PPAR-a luciferase assays
COS-7 cells (monkey kidney fibroblast-like cells) were grown in
DMEM supplemented with 10% fetal bovine serum and 1% Pen/
Strep under standard conditions. Cells were plated in a 24-well
plate at confluence and transfected using Lipofectamine LTX and
PLUS Reagent (Life Technologies 15338-100) according to the
manufacture's instruction. At day 1, for each well, a combination of
25 ng of mouse PSG5- PPAR-a (plasmid 22751; Addgene), 300 ng of
324 G. Donvito et al. / Neuropharmacology 148 (2019) 320e331
PPRE X3-TK-luc; (Plasmid 1015 Addgene), 100 ng of pSV-b-Galac-
tosidase Control Vector (Promega E1081) and 75 ng pcDNA3
(Invitrogen) empty vector to a total of 500 ng were transfected. The
following day, the growth media was replaced with fresh media
containing compounds listed below and treated overnight. Ethanol
was used as vehicle. At day 3 after 18 h of treatment cells were
harvested and processed for the Luciferase and b -Galactosidase
detection analysis.
Luciferase Gene Reporter activity was detected using the Lucif-
erase kit (Sigma, LUC1) whereas b-Galactosidase Activity was
detected with the b-Galactosidase detection kit (Sigma, Gal-A). b-
Galactosidase expression was quantified with the Microplate
Readers (Tecan) and used as an internal experimental control to
analyze the transfection efficiencies in each cell sample group. The
levels of Firefly Luciferase chemiluminescence intensity was
detected with a ChemiDoc MP system station using the Imagelab
software (Biorad) and reported normalized with respect to the b-
Gal expression.
2.13. Statistical analysis
Lipid levels are expressed as means ± SEM of pmols/g wet tissue
weight, unless otherwise stated. One-way ANOVA followed by the
Tukey's test was used for comparisons of AEA, 2-AG, PEA, OEA and
OlGly levels among the various groups. Unpaired t-test was used to
analyze the data on the distribution of OlGly in the brain.
In the nicotine withdrawal studies, the data were analyzed by
two-way ANOVA followed by Holm-Sidak's post-hoc test. For
conditioned place studies, a preference score was calculated by
subtracting time spent in the nicotine-paired chamber post-
conditioning minus the time spent pre-conditioning. A positive
value indicated a preference for the nicotine- (or morphine-) paired
compartment, whereas a negative value indicated an avoidance of
the nicotine- (or morphine-) paired compartment. A number at or
near zero indicated no preference. Data were analyzed by one-way
ANOVA and further analyzed by the Student Neuman-Keuls post-
hoc test.
In tetrad studies and luciferase assay, data were analyzed by
one-way ANOVA followed by the Dunnett's post-hoc test. In the
cumulative dose-response of CP55,940, data were analyzed by two-
way ANOVA followed by the Sidak post-hoc test. In the luciferase
assay, the Student's t-test with Welch's correction was applied. In
the binding studies, Ki values were calculated by applying the
Cheng-Prusoff equation to the IC50 values for the displacement of
the bound radioligand by increasing concentrations of the test
compound. Data are expressed as means ± SEM of at least n ¼ 3
experiments.
The computer program GraphPad Prism version 6.0 (GraphPad
Software Inc., San Diego, CA) was used in all statistical analyses. All
data are expressed as mean ± SEM. A P value of <0.05 was
considered statistically significant.
3. Results
3.1. OlGly levels are increased in the insular cortex of TBI mice
Experimental TBI increased OlGly levels, but not other analyzed
lipids, in the insular cortex of mice [F (2,10) ¼ 15.46, p < 0.001;
Fig. 1A] compared to sham and naïve mice. However, OlGly levels
did not significantly differ between brain-injured and sham groups
in the hippocampus (Fig. 1B) or hypothalamus (Fig. 1C), indicating
regional selectivity. The chromatograms show the presence of
OlGly in the insula of TBI mice (Fig. 1D), but not in sham (Fig. 1E) or
naïve (Fig. 1F) mice. Fig. 2 shows endocannabinoid levels (i.e., AEA,
2-AG) and related N-acyl-ethanolamines levels (i.e., PEA, OEA) in
each of the three brain regions. As shown in Fig. 2A, TBI elicited a
significant decrease in AEA levels in the insula [F (2,8) ¼ 19.18,
p < 0.001] and hippocampus [F (2,8) ¼ 7.587, p < 0.01]. The levels of
other investigated fatty acid amides in the examined brain regions
of each experimental group were below detection.
3.2. OlGly attenuates nicotine withdrawal and nicotine reward
Based on the mouse brain injury data revealing a region-
selective increase of OlGly in the insula, we elected to examine
whether an i.p. injection of OlGly given 15 min before the nicotine
receptor antagonist mecamylamine would prevent withdrawal
signs in nicotine-dependent mice. Whereas nicotine-dependent
mice undergoing precipitated withdrawal showed a decrease of
time spent exploring the open arm in the elevated plus maze assay,
OlGly (30 and 60 mg/kg, i.p.) significantly increased open arm time
[F (3, 56) ¼ 4.661, p < 0.01; Fig. 3A]. Moreover, 60 mg/kg OlGly
attenuated the number of mecamylamine-precipitated somatic
withdrawal signs in nicotine-dependent mice [F (3, 56) ¼ 3.439,
p < 0.01; Fig. 3B], but did not elicit overt behavioral alterations in
control mice continuously infused with saline.
Next, we evaluated whether i.p. administration of the most
effective dose of OlGly (60 mg/kg) is brain penetrant. Fifteen mi-
nutes following the injection, we detected OlGly levels in plasma
(2004 ± 626.9 pmol/ml), insula (168.0 ± 60.28 pmol/g), hippocam-
pus (186.0 ± 60.05 pmol/g), and hypothalamus (245.6 ± 108.1 pmol/
g). In contrast, OlGly levels were below the limit of quantification in
the vehicle-injected group.
3.3. OlGly attenuates nicotine-induced CPP
We employed the CPP assay, in order to test whether OlGly re-
duces the rewarding effects of nicotine. OlGly dose-dependently
prevented the development of nicotine-induced CPP [F
(7,55) ¼ 12.77, p < 0.001; Fig. 4A], but did not affect place prefer-
ence in saline-treated mice. However, the OlGly reduction of
nicotine CPP did not carry over to morphine CPP (Fig. 4B), sug-
gesting that OlGly is comparatively selective in blocking nicotine
CPP.
3.4. OlGly does not functionally interact with the endogenous
cannabinoid system
Because the endogenous cannabinoid system modulates
numerous indices of nicotine reward and dependence, the next
series of experiments investigated OlGly in a variety of in vivo and
in vitro assays indicative of cannabimimetic activity. OlGly (10, 30,
and 100 mg/kg i.p.) did not elicit significant effects in the tetrad
assay, a battery of in vivo measures highly associated with CB1 re-
ceptor activity (Martin et al., 1991). Specifically, OlGly did not elicit
catalepsy in the bar test, antinociceptive effects in the tail-flick test,
hypothermia, or hypomotility (Table 1). Additionally, OlGly did not
alter the dose-response relationship of the high efficacy CB1 re-
ceptor agonist CP55,940 (Devane et al., 1988) in the tetrad assay
(Fig. 5), suggesting that it does not act functionally as a CB1 receptor
antagonist or CB1 receptor allosteric modulator. Finally, OlGly did
not bind CB1 or CB2 receptors and weakly inhibited FAAH (Table 2).
3.5. OlGly elicits its effects via PPAR-a mechanism of action
Considering that OlGly possesses a chemical structure similar to
 G. Donvito et al. / Neuropharmacology 148 (2019) 320e331 325

Fig. 1. Traumatic brain injury (TBI) leads to increased OlGly levels in the insular cortex of mice. (A) Mice subjected to TBI display significant increases of OlGly in the insula, but not in
(B) hippocampus or (C) hypothalamus. Values represent the mean ± SEM of n ¼ 5. **p < 0.001 vs naïve; ##p < 0.001 vs sham. (D) A representative chromatogram shows the
presence of OlGly in the insula of mice 24 h following TBI. Injured insula shows formation of OlGly as confirmed by MS and MS/MS spectra. However, OlGly was below the limit of
detection in sham (E) and naïve mice (F). The arrow depicts the retention time of OlGly from the standard. The upper chromatogram traces represent the total ion current (TIC), and
lower chromatogram traces represent the extracted chromatograms m/z around 340 amu in (D-F).

OEA, which acts as an endogenous PPAR-a ligand, we next assessed
whether OlGly binds PPAR-a as well as produces functional activity
at this receptor. As shown in the modeling experiment, OlGly binds
PPAR-a (Fig. 6A), and behaved as a PPAR-a receptor agonist in a
specific luciferase assay for functional activity (Fig. 6B). The selec-
tive PPAR-a agonist GW7647 (comparison drug), as well as OlGly,
significantly increased luciferase activity compared to DMSO [F
(5,44) ¼ 17.7, p < 0.0001].
Given this in vitro evidence suggesting that OlGly behaves as a
PPAR-a receptor agonist, we next examined whether PPAR-a me-
diates OlGly prevention of nicotine CPP. As shown in Fig. 6C, the
selective PPAR-a antagonist GW6471 fully prevented OlGly
blockade of nicotine CPP [F (6, 56) ¼ 12.8, p < 0.0001].
4. Discussion
The impetus of the present study was based on a report that
nicotine addiction was ameliorated in cigarette smokers suffering
from brain damage that included the insular cortex compared with
brain-damaged individuals that did not involve the insula, sug-
gesting that this brain region may play an important role in
smoking addiction (Naqvi et al., 2014, 2007). We speculated that
326 G. Donvito et al. / Neuropharmacology 148 (2019) 320e331

Fig. 2. Levels of endocannabinoids in the hippocampus, hypothalamus and insula of sham mice and mice following weight drop-induced traumatic brain injury. (A) Levels of
anandamide (AEA) and 2-arachidonoylglycerol (2-AG). (B) Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) levels. Values represent means ± SEM of n ¼ 4e5 ex-
periments. **p < 0.001, *p < 0.05 vs naïve; ##p < 0.001, #p < 0.05 vs sham.

Fig. 3. OlGly prevents mecamylamine-precipitated withdrawal signs in nicotine-dependent mice. Intraperitoneal administration of OlGly significantly (A) increased time spent in
the open arms of the elevated plus maze compared to nicotine-pelleted mice that were given an injection of vehicle, and (B) decreased total somatic signs. All mice were challenged
with mecamylamine (2 mg/kg, s.c.) prior to testing. MP: minipump. Values represent the mean ± SEM of n ¼ 7e8 mice per group. *p < 0.05 vs. saline/vehicle; #p < 0.05 vs. nicotine/
vehicle.

experimental brain injury in mice might alter neurochemicals in
this brain region that may serve as a starting point for identifying
new molecules to treat nicotine addiction. Accordingly, we exam-
ined the lipidomic profile of the insular cortex in mice subjected to
TBI and found a profound increase of a largely uncharacterized N-
acylglycine, OlGly, in the insular cortex, but not in hippocampus or
hypothalamus. N-acylglycines represent a possible class of endog-
enous signaling molecules, which are structurally related to
endocannabinoids, and have recently generated research interest
due to their remarkable biological activities, such as anti-
inflammatory effects (Burstein et al., 2011), inhibition of cancer
cell proliferation (Chatzakos et al., 2012), adipogenesis (Wang et al.,
2015), and neural protection (Cohen-Yeshurun et al., 2011). Based
on this regionally selective increase of OlGly, we sought to test
whether its exogenous administration would ameliorate
withdrawal-associated behaviors in nicotine-dependent mice as
well prevent nicotine reward in the mouse CPP model. Three gen-
eral findings support the idea that OlGly may serve to counteract
nicotine addiction. First, OlGly reduced somatic withdrawal signs as
well as affective behavior in nicotine-dependent mice undergoing
precipitated withdrawal. Second, OlGly prevented the development
of nicotine CPP. Third, a singular systemic administration of the
most effective dose of OlGly in naïve mice results in an increase of
OlGly contents within plasma, and in the brain to levels similar to
those found in mice after TBI.
Few studies have investigated the pharmacological effects of
OlGly. Although it has been detected in different tissues, including
rat spinal cord, brain, lung, skin, ovaries, liver, spleen, and kidney
 G. Donvito et al. / Neuropharmacology 148 (2019) 320e331 327

Fig. 4. OlGly prevents nicotine reward, but not morphine reward. (A) OlGly dose-dependently blocked nicotine CPP, (B) but did not attenuate morphine (30 mg/kg, i.p.) CPP. Values
represent the mean ± SEM of n ¼ 7e8 mice per group. *p < 0.05 vs. saline/vehicle; #p < 0.05 vs. nicotine/vehicle.

Table 1
OlGly does not elicit in vivo pharmacological effects as assessed in the tetrad assay. OlGly did not produce catalepsy, antinociception, hypothermia, or motor behavior, as
reflected by the following measures: distance traveled, speed, and immobility time. Values represent means ± SEM of n ¼ 9 mice per group.

Treatment Catalepsy (s) Antinociception (%MPE) D rectal temperature (
C) Distance traveled (cm) Speed (cm/s) Time immobile (s)

vehicle 0.0 6.0 ± 5.1 0.41 ± 0.30 20.8 ± 2.3 6.9 ± 0.8 7.1 ± 1.5
OlGly 10 mg/kg 0.0 12.0 ± 6.6 0.90 ± 0.19 24.3 ± 1.2 8.1 ± 0.4 8.6 ± 1.6
OlGly 30 mg/kg 0.0 6.0 ± 4.5 1.13 ± 0.26 21.5 ± 1.7 7.2 ± 0.6 11.8 ± 6.2
OlGly 100 mg/kg 0.0 25.9 ± 11.0 0.22 ± 0.5431 18.6 ± 1.5 6.21 ± 0.5 17.4 ± 7.3

Fig. 5. OlGly does not act as a CB1 receptor allosteric modulator in the in vivo triad assay. CP55,940 (0, 0.3, 1, and 3 mg/kg) dose-dependently induced (A) catalepsy, (B) anti-
nociception, and (C) hypothermia in vehicle-pretreated mice. Pretreatment with OlGly (60 mg/kg) did not induce any changes in the dose-response curve of CP55,940 for any of
these dependent measures. Values represent means ± SEM of n ¼ 5/6 mice per group.

Table 2
OlGly does not functionally interact with the endogenous cannabinoid system. OlGly shows low affinity for CB1 and CB2 receptors and has very low potency in inhibiting FAAH.
Values represent mean ± SEM (n ¼ 3), % displacement or inhibition, n ¼ 2.

CB1 IC50 CB1 Ki CB1 displacement produced by CB2 IC50 CB2 Ki CB2 displacement produced by IC50 FAAH FAAH inhibition produced by
(mM) (mM) maximum dose tested (50 mM) (mM) (mM) maximum dose tested (50 mM) inhibition (mM) maximum dose tested (50 mM)

45.5 ± 5.1 27.8 ± 3.1 55.9 ± 7.3% >50 >50 15.5 ± 6.40% 8.65 89.4%

(Bradshaw et al., 2009), the present study is the first to quantify it in
specific brain regions. OlGly biosynthesis results from a reaction
between oleoyl CoA and glycine, which can be catalysed by either
glycine N-acyltransferase-like 3, or cytochrome c in the presence of
hydrogen peroxide (Wang et al., 2015), while FAAH participates in
its degradation (Bradshaw et al., 2009). Additionally, OlGly may
play a role in the biosynthesis of oleamide, another substrate of
FAAH that mediates several fundamental neurochemical processes
(Boger et al., 1998) including sleep (Huitron-Resendiz et al., 2004),
thermoregulation, and nociception (Fedorova et al., 2001). OlGly
and oleamide possess equal potency in eliciting hypothermia and
decreasing locomotion in rats. However, the failure of OlGly to in-
crease circulating levels of oleamide suggests that its
pharmacological effects occur independently of its conversion to
oleamide. In addition to its structural similarity to the N-acyle-
thanolamines, which include AEA, as well as PPAR-a agonists OEA
and palmitoylethanolamide (PEA), recent evidence suggests that
OlGly may play a role in enhancing insulin sensitivity through a CB1
receptor mechanism of action (Wang et al., 2015). The CB1 receptor
also plays an important role in mediating the rewarding effects of
nicotine. For example, a CB1 receptor antagonist administered
either systemically or into the ventral tegmental area (VTA)
decreased nicotine self-administration in rats (Le Foll and Goldberg,
2004; Simonnet et al., 2013). Similarly, CB1 (/) mice or wild type
animals treated with a selective CB1 receptor antagonist do not
display nicotine CPP (Castan~e et al., 2002; Le Foll and Goldberg,
328 G. Donvito et al. / Neuropharmacology 148 (2019) 320e331

Fig. 6. PPAR-a mediates the anti-reward effects of OlGly. (A) Representative frame from MD of OlGly/PPAR-a complex and details of ligand-protein interactions in the ligand binding
site. The carboxylic moiety of the ligand recapitulates the main polar stabilizing interactions with Tyr464(H12), Tyr314(H5), His440(H10/11) and Ser280(H3), signature of a PPAR-a
agonist. (B) Luciferase Assay for PPAR-a/RXR. Relative Luciferase Units in response to OlGly and the PPAR-a agonist GW7647 (comparison drug). DMSO (n ¼ 12), GW7647 (GW)
10 mM (n ¼ 6), OlGly 10 mM (n ¼ 7), 50 mM (n ¼ 9) and 100 mM (n ¼ 11). ****p < 0.0001, *p < 0.05 vs DMSO (C) The selective PPAR-a antagonist GW6471 (2 mg/kg) prevented OlGly-
induced inhibition of nicotine CPP. n ¼ 6e8 mice per group. ****p < 0.0001. ***p < 0.001, **p < 0.01 vs. vehicle/vehicle; ###p < 0.001 vs. nicotine/vehicle. Values represent the
mean ± SEM.

2004; Merritt et al., 2008), indicating that this receptor plays a
necessary role in multiple laboratory models of nicotine reward.
Likewise, genetic deletion or pharmacological inhibition of CB2
receptors blocks nicotine reward-like effects in the nicotine self-
administration and nicotine CPP paradigms in mice (Ignatowska-
Jankowska et al., 2013; Navarrete et al., 2013). In contrast, a CB2
receptor antagonist did not affect either nicotine self-
administration or reinstatement of nicotine seeking in rats
(Gamaleddin et al., 2012). These divergent results of the effective-
ness of CB2 receptor antagonists in reducing nicotine reward may
be related to species differences. Importantly, clinical trials
revealed the CB1 receptor antagonist rimonabant effectively
improved smoking abstinence compared with placebo, though
clinical development of this and other CB1 receptor antagonists was
terminated due to unacceptable adverse effects (Robinson et al.,
2017). The question of whether a CB2 receptor antagonist has effi-
cacy in treating smoking addiction remains to be determined.
Based on the extensive role that the endogenous cannabinoid
system plays on nicotine reward, we tested whether OlGly interacts
with this system. OlGly did not produce in vivo pharmacological
activity indicative of CB1 receptor stimulation as assessed in the
tetrad assay, consisting of catalepsy, antinociception, hypothermia,
and hypomotility measures (Little et al., 1988; Martin et al., 1991;
Wiley and Martin, 2003). In contrast, Chaturvedi and colleagues
reported that OlGly reduces body temperature (1.5e2.0
C) and
decreases locomotor activity in rats (Chaturvedi et al., 2006). The
disparate results between their finds and the results in the present
study may be a consequence of species differences or other
methodological considerations (e.g., housing of the animals,
ambient room temperature, etc.). At any rate, the previous study
did not examine CB1 receptor involvement of the hypothermic or
decreases locomotor activity effects of OlGly. Furthermore, the fact
that OlGly poorly binds human recombinant CB1 and CB2 receptors
suggests direct action at these receptors is unlikely. Another pos-
sibility is that OlGly behaves as an allosteric modulator of the CB1
receptor. Accordingly, we examined whether OlGly would alter the
antinociceptive, cataleptic, and hypothermic effects of the high
efficacy, orthosteric CB1 receptor agonist CP55,940. Whereas the
CB1 receptor positive allosteric modulator ZCZ011 elicited a left-
ward shift of the CP55,940 dose-response curve for each of these
measures (Ignatowska-Jankowska et al., 2015), OlGly did not affect
the CP55,940 dose-response curves. It is noteworthy that FAAH
inhibitors reduce nicotine self-administration and nicotine
priming-induced reinstatement through PPAR-a in squirrel mon-
keys, consistent with the idea that the endogenous substrates of
this enzyme counteract nicotine reward by activating this nuclear
receptor (Justinova et al., 2015). In contrast, Forget and colleagues
have found that inhibition of FAAH reduced nicotine reinstatement,
but did not affect nicotine self-administration (Forget et al., 2009).
Accordingly, our experiment showing that OlGly only weakly in-
hibits AEA hydrolysis suggests the unlikelihood of indirect activa-
tion of cannabinoid receptors or other AEA targets through FAAH
inhibition.
Structural similarities between OlGly and OEA, which activates
PPAR-a, raise the possibility that OlGly may likewise prevent the
development of nicotine CPP through this receptor. Notably, PPAR-a
has been implicated in the control of nicotine reward (Jackson et al.,
2017; Mascia et al., 2011; Panlilio et al., 2012). The modeling
experiment presented here revealing that OlGly binds PPAR-a and
the finding that OlGly behaved as a PPAR-a agonist in a specific
luciferase assay support this hypothesis. Another noteworthy
finding in the present study is that the selective PPAR-a antagonist
GW6471 fully prevented OlGly blockade of nicotine CPP, indicating
that this anti-reward effect of OlGly requires PPAR-a receptor
activation. These results are consistent with recent reports on
PPAR-a agonists blocking nicotine withdrawal, nicotine CPP, nico-
tine self-administration, and nicotine priming-induced reinstate-
ment in rodents and nonhuman primates (Jackson et al., 2017;
Mascia et al., 2011; Panlilio et al., 2012). However, fenofibrate, a low
efficacy and non-selective PPAR-a agonist, did not facilitate the
ability to stop smoking during a brief practice quit period in
dependent smokers (Perkins et al., 2016). In addition, it failed to
reverse nicotine withdrawal in mice (Jackson et al., 2017). None-
theless, clinical studies employing PPAR-a agonists with higher
efficacy and selectivity remain to be conducted.
The findings that OlGly dose-dependently reduced the devel-
opment of nicotine-induced CPP, but did not affect morphine CPP,
suggest selectivity for blocking nicotine CPP. This observation is
consistent with work reviewed by Melis and Pistis (2014), who
reported that the “anti-addictive” properties of PPAR-a stimulation
show selectivity for reducing nicotine reward over the reinforcing
effects of cocaine, cannabinoids, and morphine (Melis and Pistis,
2014). A parsimonious explanation accounting for the selectivity
of OlGly in blocking nicotine CPP includes a complex
 G. Donvito et al. / Neuropharmacology 148 (2019) 320e331
neurochemical pathway involving PPAR-a activity inducing nega-
tive modulation of b2*-nAChR receptor activity expressed on VTA
dopamine cells that ultimately dampen nicotine-induced firing and
bursting activity (Melis et al., 2013). Similarly, fenofibrate induced a
reduction of stress-induced depression behaviors in mice through
phasic activation of the mesolimbic dopaminergic system (Scheggi
et al., 2016). Additionally, it has been reported that local infusion of
dopamine receptor 1 antagonist into the insular cortex significantly
decreased nicotine self-administration in rats (Kutlu et al., 2013).
According to the model proposed by Naqvi and Bechera, the
insula belongs to a network, which includes the ventromedial
prefrontal cortex, anterior cingulate cortex, NAc, and amygdala,
responsible for the conscious pleasure from drugs, drug craving,
drug seeking behavior, and drug relapse. Specifically, the insula
processes interoceptive information about the drugs received from
a thalamocortical pathway and relays this information to the pre-
frontal regions and the amygdala, which evoke pleasure related
effects of the drugs through dopaminergic activation in the VTA
(Naqvi and Bechara, 2010). According to their model, the insula
serves as a critical neural substrate for addiction by regulating the
dopaminergic signal in the VTA. However, whether a direct
connection exists between the mesolimbic dopamine system and
PPAR-aeinduced reduction of nicotine CPP by OlGly remains an
unanswered question. It will also be important to determine
whether the anti-nicotine CPP effects of OlGly extend to self-
administration and reinstatement procedures.
We based the rationale to investigate the effects of OlGly on
nicotine CPP and withdrawal responses in nicotine-dependent
mice from lipidomic analyses of the insular cortex from mice
following TBI. Although the present study does not address the
neural substrates underlying these effects, we found that following
a singular i.p. administration of a dose of OlGly that was most
effective in reducing nicotine withdrawal, the levels of OlGly
increased within the insula, hippocampus, hypothalamus and
plasma up to concentrations that were similar to those detected
after TBI. Nonetheless, other preclinical research supports a role of
the insular cortex on nicotine reward. Whereas inactivation of
insula by local infusion of geaminobutyric acid agonist mixture
decreased nicotine self-administration in rats (Forget et al., 2010;
Pushparaj et al., 2015), electrical stimulation of this region signifi-
cantly attenuated nicotine reinforcement as well as nicotine-
seeking behaviors. These findings suggest that the insular cortex
may play a modulatory role in nicotine reward (Pushparaj et al.,
2013). In the case of dependence, both central and peripheral
nicotinic receptors mediate somatic nicotine withdrawal signs,
while central nicotinic receptor populations, such as the nucleus
accumbens (NAc) and habenulo-interpeduncular system regulate
anhedonia, anxiety, and aversion (De Biasi and Dani, 2011; Watkins
et al., 2000). Furthermore, the mesohabenular pathway through
the interpeduncular nucleus and corticotropin-releasing factor-1
(CRF1) receptors in the central nucleus of the amygdala (CeA) seem
to play a preponderant role in the emergence of anxiety-like be-
haviors in nicotine-dependent animals (Cohen et al., 2015). Thus,
the neural sites of action of OlGly and whether the elevated levels of
OlGly in the insular cortex produced by TBI ameliorates nicotine
reward and/or dependence remain to be determined.
5. Conclusions
The present study demonstrates an alteration in the lipid profile
in the insular cortex compared to other neural regions of mice
subjected to TBI. In particular, we found a significant increase of
OlGly levels in the insula, a region implicated in smoking addiction
(Naqvi et al., 2014, 2007). A potential PPAR-a mechanism of action
is consistent with several reports showing that PPAR-a agonists
329
attenuate nicotine reinforcement and reinstatement in rodents and
nonhuman primates (Mascia et al., 2011; Panlilio et al., 2012),
though the neuroanatomical substrates remain to be determined as
well as the potential translational efficacy in humans. Nonetheless,
the findings that i.p. administration of OlGly prevented both nico-
tine CPP and nicotine withdrawal-associated behaviors in mice, and
that the injection of the same effective dose of OlGly in naïve mice
was brain penetrant suggest that this ligand may be of value in
treating addiction to tobacco smoking in humans.
Funding
This work was supported by NIH grants P01DA009789,
R01DA039942, P30DA033934 (AHL); R01 DA032246 and
P50DA039841 (MID), T32DA007027 (AJ), and startup funds from
the VCU School of Pharmacy. NSERC (92056) and CIHR (137122)
grants to LAP.
Conflicts of interest
None.
Authorship contributions
Participated in the research design: Donvito, Piscitelli, Petrie,
Parker, Maione, Lichtman, Damaj, Di Marzo, Mechoulam.
Conducted experiments: Piscitelli, Muldoon, Jackson, Vitale,
D'Aniello, Giordano, Guida, Ignatowska-Jankowska, Mustafa, Sim-
Selley.
Contributed to reagents or analytical tool: Smoum.
Performed data analysis: Donvito, Piscitelli, Muldoon, Jackson,
D'Aniello, Mustafa, Lichtman, Damaj, Di Marzo.
Wrote or contributed to the writing of the manuscript: Donvito,
Piscitelli, Lichtman, Damaj, Di Marzo, Mechoulam.
Declarations of interest
Drs. Damaj, Di Marzo, Lichtman, Mechoulam, and Parker filed a
provisional patent on oleoyl glycine.
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